Method of accelerating propionic fermentation



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Patented Dec. 5, 1933 UNITED ls.*TAIKYE-SA Pi-f The Wilbur Owego, N. Y.;

White Chemical Company,

a corporation of New York No Drawing. Application April 25, A1928 I `Serial No. 212,863

l' 2- olaims. i (c1. 26o- 420) This invention relates to processes for producing propionic acid or` propionates from materials' containing carbohydrates and/or salts of certain organic acidsby the, fermentation-of the said materials using organisms of the type lof Bac- 'terum acidi-grwopz'o1tici,A and more particularly resides in methods of increasing the intensity and rate of the propionic fermentation.

1 While it is, of course, well known that carbo- A hydrate-containing.materials and/ or salts of or# ganic acida-'such as, for example, molasses or other fermentable carbohydrates, or salts of lactic acid-may be caused to, undergo propionic fermentation by means of organisms-of the'type of Bacteriu'm acidi-propionici, it is equally Well known that such fermentations heretofore have not .been reduced 'to commercial-practice to any large extent. There appear to .be-two complementary explanations therefor, to wit: (1) 'The relatively slow. growth of the-individualscells and 4 they limited accumulation (i. e., relatively low numerical strength) of lthe same attained .in `za normal growth cycle 'of a`culture in aunit volume of suitable medium, and `(2) the characteristic, peculiar to organisms ofthe type mentioned, of a relatively long period of lag of unproduc tive dormancy in the growth cycle of the organ# vWhen a suitable sterilemediumis inoculated with an-old culture of the organisms, a definite growthcycle may be observed: I. At the'beginning'there is. a-period Aof. lag or dormancy during'which no Vreproduction-"-takes place. During this period the olldcellsundergo a process of rejuvenation whichV` prepares themlfor reproduction.

. II.v After the lag phase, the-culture undergoes rapid growth during which the "numberv of organisms approximately doubles at the end of each generation time. This period' of the cycle is known as the logarithmic growth phase,-''it is duringthis phase that the'individual cell evidences the greatest fermentativeY activity. l

IIL-,Following-the logarithmic growth phase, the number of organisms remains fairly constant, or increases slowly, for anadditional period.

IV. Finally, a period is reached during which the' death rate ismore rapid thanthe'rate of reproduction and the number of living cells constantly decreases. y

The above growth cycle is not peculiar to any one typeof fermentation organism;'- however, in most-microbic .fermentations the period of lag is so short and the growth or reproduction rate is so rapid as tomake it unnecessary.' in, prac- An object of `the-'present invention, therefore,

tical applications toconsider thesephases of they vlife cycle. In `the'caserof propionic fermentation,`

on the other hand, the lag phaseof organisms of the type of Bacterium acidi-p'ro'piomcH is, markedly long, andltheir. reproduction rate.; is slow, hence, these phases areof particular con-l cern since they probably accountfor4 the., non-1 usev of propionic fermentation inthe industrialart. .Y

Again, it-is welliknown that, in ymicrobic fer-A mentations, the products thereof do not appear in significant quantities until after the organisms have undergone extensive reproduction and have attained Vapproximately their maximumnumf bers.' This-is because the amount of fermentation product vproduced. by the individual cell is so small that itcan be measured chemically only after enormous numbers oforganisms have been produced and their fermentation products have accumulated.

is to .provide a procedure whereby the' number of propionic fermentation )organisms in o a ygiven amount of fermentativemedium may bearti-` ficially' increased overjthat, obtainable'by a nor'- mal fermentation. i" I f Another object is to providea'method of in! creasing the'intensityand speed-of the propionic` fermentation' by -maintainingthe organisms in a state of-,vphysiological youth. A

It hasA nowbeen found that the great mass of cells necessary for rapid propionic fermentation may be "obtained by propagating `the organismsy undery such v,conditions as i. make Ypossible thevvith-Y drawal of fermented medium and the addition of: fresh medium, Vwith the retention of al large profl portion of the cells, during the'fermentation op-A eration.- The -mass of retained cellsserves as a mass inoculum vfor the succeeding charge of fei'rmentable material (i. e., the -r`rledium),v thus' causing the-fermentation to start l quickly and proceed rapidly. r-As this process is continued or repeated,

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'there' is accumulated a'mass of min crobQ-cells which far exceeds in numbers thatobtained/"when a culture ofthelsame organisms 1' goes throughits vnormal growth cyclein an une changedmedium of the same composition. VWith f 4increase of 'the 'mass-of cells there is ai correthe rate of, fermentationi- This invention, then, is based upon theflcon-A ception ci. artificially increasing th'e'mass ofv pro; -pionic organisms beyond that 4spontane ously developed in a'givengbody of medium, fermenting suitable medium ,in Contact with this 'increased mas-sof cells, vseparating at least the major por'.

spending increase in NTgoFFlcE-f los cells.

tion of the mass from the fermented medium and fermenting a fresh charge of medium withvthe separated mass of cells.

may be effected by any one of Thus, for example, VI have This method several procedures. found that one operative procedure consists 'in carrying out the fermentation in a chamber or container in which a finely divided or porousinert substanca-sucha's limestone, sand, cinders, charcoal, or the likeis suspended, or in which the said substance covers the bottom. This finely divided or porous substance furnishes surfaces on which the bacteria grow' and to which they tend toV adhere when the fermented liquid is removed. Another operable procedure consists in carrying out the fermentation Ain a tower, column or bed composed of, or in which are interposed a plurality of beds of, nely divided or porous inert substances on thevsurface of which the cells grow. Thev fermentable medium may be led through the tower, columnv or bed, and into and out ofcontact with the bacteria-encrusted substances ffilling the same, vwith minimum loss of cells. Furthermore, a :lter may be interposed betweenthe mass of cells and the outlet of the fer- I mentation chamber r`or container, for the purposeof retaining all or part of the ,cells when fermented liquid is withdrawn. Substantially the sameieifect may be produced by absorbing' the organisms'and their .enzymes in a suitable solid adsorbent,v such as, the material known as Filter-Cel, or other argillaceous or diatomaceous material, and bringing aboutV the fermenl tationof fermentable mediumvin contacttherewith. y

I have found, furthen'that the intensity and speedzof the propionic fermentation'may be increasedby effecting the removal of fermented medium and introduction of fresh charge of fer-A youth, and thus still further increase `thefermenting capacity of the artificially increased Vmass of The in'vention'will be more specically Y described by recourse to the following specific eX- amples, it being understood that the-invention is not restricted to the particular means, ma-

jterials, procedures or conditions therein set out.

Eample 1.-Into an open lor closedvesrsel-there is introduced 1 part by weight of a -fermentable medium consisting of v molassesin water (i. e., equivalent tor an aqueous solution of and containing l percent, more or less, by weight of meat scrap.. There are then added, infany order, infusorial earth A.in such amount that theearth occupiesabout 1/3 .to 1 /2 of the volume of the contents ofthe vessel, and calcium carbonate in amountat least equal to percent or'more ofy propionic and Y l Y, .usually in about the proportion of Y 2 Vparts lof' That is to say, I coordinatewith the it is possible to maintain" the or-v ganisms in -ap state Yof constant physiological a 10 percent Ysolution of sugar of about 5 `percent strength.)

1/2 the weight of the` carbohydratecontent of the medium.'` The mixture is then sterilized Yand there is then" added a culture comprising Bacterz'am acidi-propionicz' and Lacto-bacillas casei. f Fermentation is allowed to proceed at a temperature offrom about 30. to 35 C. for about 4 days, preferably, intermittent mild yagitation is carried out during the first 3 days with settling of thesolids during the 4th day. The supernatant liquid is then withdrawn and is replaced b y an equivalent amount of fresh sterile medium. The above cycle is repeated, the fermentation becoming .more vigorous with each successive repetition until the maximum yield of propionic and acetic acids is attained. This point is reached usually at about the 4th repetition of the cycle.

The cyclic procedure may be continued inde- Vnitely, fermented liquid. being vnthdrawn and a fresh charge of fermentable medium being addedy about every 4rdays.

Under the conditions described,vit has been established that 'a 3 percent `yield be obtained in from'2 to 4days. the fermented liquid is found to contain about f3 acetic acids propionic acid to 1 part of acetic acid. y

It is to be-understood'that, in the-above procedure, I may-substitute for the molassesan equivalent amount of 4a sugar or of any other fermentable carbohydrate material, f and 'fforl the meat scrap I may substitute'anysuitableorganic source of nitrogen such as protein materials generally, dried yeast, blood `mealyor the like.

conditions above set out,` it now seems desirable tocarry on the fermentation operation at a temperature of about 30 to 35 C., it is'possible to effect the fermentation under other temperature conditions. Thus, it hasbeen foundA possible to effect the fermentation at temperatures from as low as`15 C. or somewhat'lower, to about-40fC- or somewhat'higher.

or better canV That -is to say,

Itis also torbe understood that whileglunderthe Example 2.*Acc`ording -to the-'procedure set Y out in Example 1, an aqueous-solutionof calcium lactatecontaining calcium lactate equivalent to `5 percent of lacticacid is fermented using-as lfer- Vmenting means a 'cultureV comprising Bacterzumacz'clz propionicz' and Lactobacillus casei.

In fermenting an aqueous solution of calcium lactate it is to-.be understood that the vpresence of lactic acid bacteriaA is not essential: fTheirpr es` ence, however,- lhas been found to effect'aV desirable stimulation of the activity ofthe Bacterniam acidi-propionicz'. Insteadl of Lactobacillus casei,l

'there maybe used other-.organisms evidencing a desirable stimulation of the activityf of the -p'ropionic acidfproducing organisms, asldescribed in U.S. Patent No. 1,459,959.

j As described inf-theSherman et al...Patent fNo. 1,470,885, it appearsfto be economicallydesirable to first convert the carbohydrate content of earbohydrate containing materials undergoing propionicfermentation into lactates and to Yfbring about the propionic fermentation ofthe vresulting lactates.l f f Icl'aimz'v 1. In processes for the productioniof propionic v acid compounds from carbohydrates or other suitable fermentable media y.by the fermentative ction of organisms of the type ofvBactertam acidi-propionici, Vthe steps which consist in` propl agating a mass ofthe organisms von the surfacesofnely divided particles of. inert solid absorp- Y tive material whereby to provide for ready Yseparation of theorganisms andl fermented' medium,

fermenting the medium in the presence of the said mass of organisms, withdrawing at least the major portion ofthe fermented medium from Contact with the mass of organisms, and supplying fresh medium to the said mass during the fermentation operation, the withdrawal of fermented medium fromA and the supply of fermentable medium to the mass of organisms being effected during'that period of the growth cycle of the organisms which follows the dormancy phase but precedes the period during which the death rate is more rapid than the reproduction rate, and before the fermentation has gone to completion.

2. In processes for the production of propionic acid compounds from carbohydrates or other suitable fermentable media by the fermentative action of organisms acdi-propionici, the steps which consist in propagating a massof the organisms on the surfaces of finely divided particles of inert solid material, fermenting the medium in the presence ofl the of the type of Bacteriuml said mass, withdrawing at least the major portion'of the resulting fermented medium, and4 bringing a fresh supply of medium into fer-` mentative action with `the said mass during the fermentation operation, thev fermented medium being withdrawn from and the fresh medium being brought into contact with the mass of organisms during the period in ywhich the organisms are undergoing the logarithmic growth phase, and before the fermentation has gone to completion.

JAMES M. SHERMAN. 

